Polyvalent Viral Vectors and a System for Production Thereof

ABSTRACT

A system for generating viral vectors carrying two distinct expression cassettes is provided. The system utilizes a unique polyvalent transfer vector that permits efficient detection and selection of inserted expression cassettes.

BACKGROUND OF THE INVENTION

The use of viral vectors to express antigens has been described.Further, the use of fusion peptides as antigen has been described.

Fusion peptide based vectors simplify the dosing regimen and create moreopportunities for heterologous boosting. However, the unpredictablenature of fusion peptide processing and epitope presentation, anddifficulties in creating and propagating adenoviruses carrying largeinserts have become roadblocks to their large-scale applications.

What is needed are predictable methods for generating viral vectorsuseful for delivery of gene products.

SUMMARY OF THE INVENTION

A system for generating viral vectors carrying at least two distinctexpression cassettes is provided. The system utilizes a uniquepolyvalent plasmid backbone that permits efficient detection andselection of inserted expression cassettes.

Also provided are methods of generating polyvalent viral particles usingthe polyvalent backbones of the invention.

These and other embodiments and advantages of the invention aredescribed in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the assembly of a ΔE₁-ΔE₃-adenoviral vectors.

FIG. 2 illustrates the introduction of antigens into the E1 and E3deletion loci of the adenoviral vectors.

FIG. 3 is a bar chart providing the results of an in vivo comparison ofexpression from a recombinant chimpanzee C9 adenovirus expressing thesame transgene (α1AT) from the E1 locus only (dark bars), or from boththe E1 and E3 loci (light bars).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a system for generating a viral vectorcarrying multiple expression cassettes to a target. The system utilizesa DNA molecule carrying a viral genome containing movable cloningcassettes carrying marker genes. This DNA molecule is used to generate atransfer vector carrying a viral genome that contains multipleheterologous expression cassettes located in different loci within theviral genome. The viral genome carrying the heterologous expressioncassettes are rescued from the transfer vector of the invention andpackaged in a suitable viral capsid or envelope protein to producepolyvalent viral vectors.

As used herein, the DNA molecule and/or transfer vector can be derivedfrom any genetic element that can carry the viral genome according tothe invention and that is capable of transferring the genome into a hostcell. Any suitable genetic element (or backbone) can be selected,including, e.g., a plasmid, phage, transposon, cosmid, episome, and thelike. In one embodiment, the genetic element is suitable for prokaryoticexpression, although other cloning systems can be utilized.

As used herein, the term “different loci” indicates that theheterologous expression cassette for a first selected target product islocated in the viral backbone in a site that is non-contiguous with asecond heterologous expression cassette, i.e., viral sequences arelocated between heterologous expression cassettes. These loci may be indifferent gene regions or in different open reading frames within asingle gene region. Alternatively, multiple loci may be within a singleopen reading frame but non-contiguous with one another, e.g., separatedby spacers, native sequences, restriction enzyme sites, or the like.

As used herein an “expression cassette” comprises a nucleic acidsequence encoding a product for delivery to a host cell. The nucleicacid sequence encoding the product is under the control of regulatorycontrol sequences which direct expression of the product in the host.Suitably, the expression cassette is heterologous to the vectorsequences flanking the cassette. In one embodiment, the regulatorycontrol elements in each heterologous expression cassette differ fromthe regulatory control elements in the other heterologous expressioncassettes in order to minimize (or eliminate) the risk of homologousrecombination during the cloning process and in the viral manipulationprocess in cells. In one embodiment, each heterologous expressioncassette is provided with different promoters and/or enhancers, and/orpoly A sequences. However, in other embodiments, one heterologousexpression cassette in a polyvalent vector of the invention can have oneor more regulatory control element in common with another heterologousexpression cassette in the polyvalent vector. In such an embodiment, theregulatory control element is preferably a short sequence, which doesnot enable recombination.

As described herein, the encoded product may provide a target for theimmune system, in order to induce a humoral and/or cellular immuneresponse, may be an adjuvant for another encoded product, may provide animmune modulator effect, and/or may provide a therapeutic effect.Combinations of such products can be delivered in a polyvalent viralvector according to the invention.

The term “functionally deleted” or “functional deletion” means that asufficient amount of the gene region is removed or otherwise damaged,e.g., by mutation or modification, so that the gene region is no longercapable of producing functional products of gene expression. If desired,the entire gene region may be removed. Other suitable sites for genedisruption or deletion are discussed elsewhere in the application.

I. Polyvalent Viral Construct

A. A DNA Molecule Carrying a Viral Genome and Multiple Reporter Genes

In one aspect, the present invention provides a DNA molecule carryingsequences of a virus which is to be packaged into a polyvalent viralvector. In one embodiment, such a DNA molecule is a plasmid. However,another suitable genetic element, as defined above, may be selected.Introduction of the vector into a cell can be achieved by any meansknown in the art or as disclosed herein, including transfection.

The viral sequences are selected from the types of virus(es) that aredesired for use as a delivery vehicle and that have sufficient space toaccommodate multiple expression cassettes. These viral sequences can bereadily selected from among viruses having a capsid protein, e.g.,adenovirus, or from enveloped viruses, e.g., retroviruses such as felineleukemia virus (FeLV), HTLVI and HTLVII], and lentivirinae [e.g., humanimmunodeficiency virus (HIV), simian immunodeficiency virus (SIV),feline immunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal)]), poxviruses (e.g., canarypox), among others. Still otherviruses can be readily selected by one of skill in the art.

In one embodiment, the viral sequences are from an adenovirus. Suitably,the polyvalent DNA molecule contains nucleic acid sequences from anadenoviral genome that contains at least the sequences needed to packagethe viral genome into a capsid. Typically, a polyvalent adenoviralmolecule will contain the 5′ adenoviral cis-elements and 3′ adenoviralcis-elements at the extreme 5′ and 3′ termini of the adenoviral genome,respectively. The 5′ end of the adenoviral genome contains the 5′cis-elements necessary for packaging and replication; i.e., the 5′inverted terminal repeat (ITR) sequences (which functions as origins ofreplication) and the 5′ packaging enhancer domains (that containsequences necessary for packaging linear Ad genomes and enhancerelements for the E1 promoter). The 3′ end of the adenoviral genomeincludes the 3′ cis-elements (including the ITRs) necessary forpackaging and encapsidation.

In addition, the polyvalent DNA molecule may contain additionaladenoviral sequences, or may be at least functionally deleted in one ormore adenoviral gene regions. In one embodiment, an adenoviral vectorused in the invention will contain the E2 region or a functional portionthereof (e.g., the region encoding E2a and/or E2b), and one or more ofthe late genes, e.g., L1, L2, L3, L4 and L5. In some embodiments, theadenovirus vectors used in the invention may contain all or a portion ofthe E4 region (e.g., the E4 ORF6).

For example, all or a portion of the adenovirus delayed early gene E3may be eliminated from the adenovirus sequence which forms a part of thevector. The function of simian E3 is believed to be irrelevant to thefunction and production of the recombinant virus particle.

For example, an E1-deleted Ad vector can be constructed having adeletion of at least the ORF6 region of the E4 gene, or because of theredundancy in the function of this region, the entire E4 region. Stillanother vector of this invention contains a deletion in the delayedearly gene E2a. Suitably, these vectors retain the late genes (i.e., L1,L2, L3, L4, and L5), and other elements essential for packaging ofadenoviral vectors into viral particles. Deletions may also be made inthe intermediate genes IX and IVa₂ for some purposes. Other deletionsmay be made in the other structural or non-structural adenovirus genes.The above discussed deletions may be used individually, i.e., anadenovirus sequence for use in the present invention may containdeletions in only a single region. Alternatively, deletions of entiregenes or portions thereof effective to destroy their biological activitymay be used in any combination. For example, in one exemplary vector,the adenovirus sequence may have deletions of the E1 genes and the E4gene, or the E1 genes, with or without deletion of E3, and so on.

In another embodiment, a lentiviral genome is utilized. Typically, alentiviral vector plasmid contains cis-acting genetic sequencesnecessary for the vector to infect the target cell and for transfer ofthe heterologous expression cassettes. Suitably, the original envelopproteins, and gag sequence promoter have been removed.

The viral sequences in the plasmid backbone need not be limited tosequences by the type of capsid or envelope in which they are inserted.Thus, the plasmid backbone may contain viral sequences from one viralsource that are encapsidated or packaged into an envelope from anothersource. For example, a polyvalent HIV vector can be packaged into an FIVenvelope; a polyvalent FIV vector can be packaged into an HIV envelope;a polyvalent adenoviral vector can be packaged into a capsid fromanother serotype. Still other pseudotyped viral vectors will be readilyapparent to one of skill in the art.

Once the viral sequences are cloned into a plasmid using techniquesknown to those of skill in the art, the viral genome is altered tocontain a first movable cassette located in a first deleted region of aviral genome and a second movable cassette located in a second deletedregion of a viral genome. Optionally, the plasmid can contain multiplemovable cassettes, each located in a distinct locus of the viral genome.Each of the movable cassettes is flanked by a unique set of restrictionenzyme sites which permits their selective removal from the plasmid andready insertion of a heterologous expression cassette.

Each the movable cassettes used in the invention contains nucleic acidsequences of a detectable reporter gene operably linked to sequencesthat will direct expression thereof in a host cell. Suitably, each ofthe movable cassettes contains a unique reporter gene that is readilydistinguishable from reporter genes carried by other movable cassettescarried by the plasmid backbone. In one embodiment, the reporter genesexpress products which are differentiated from one another by color.

Suitable reporter genes include those encoding products which can bedistinguished from other reporter genes carried by the polyvalentplasmid backbone of the invention. For example, fluorescent proteins aredistinguishable by color upon excitation with the appropriate wavelengthof light, including, e.g., red fluorescent protein, green fluorescentprotein, blue fluorescent protein, cyan fluorescent protein,yellow-green fluorescent protein. Suitable fluorescent proteins for usefor the selected host cell type are available commercially, e.g., fromClonTech. Still other suitable reporter genes which are distinguishableby color include, e.g., gusA (blue); DsRed (red); luciferase (red); andbeta-galactosidase. Alternatively, one of skill in the art can provideanother reporter gene that is provided with a tag or label, many ofwhich are known to those of skill in the art.

Suitable reporter genes are selected with a view to the host cell systemused for cloning. The host cell itself may be selected from anybiological organism, including prokaryotic (e.g., bacterial) cells, andeukaryotic cells, including, insect cells, yeast cells and mammaliancells, as described in more detail below.

In one particularly desirable embodiment, a prokaryotic system is used.Further, the host cell is capable of transfection of DNA and expressionof the transfected DNA, and of expressing the selected reporter gene inthe manner which is desired, e.g., calorimetrically.

Examples of suitable prokaryotic systems are well known, includingbacterial cells. For example, suitable bacterial strains may include,e.g., Escherichia coli C600-F-, e14, mcrA, thr-1 supE44, thi-1, leuB6,lacY1, tonA21, [[lambda]][−] [Huynh, Young, and Davis (1985) DNACloning, Vol. 1, 56-110]; DH1-F[−], recA1, endA1, gyrA96, thi-1, hsdR17(rk[−], mk[+], supE44, relA1, [[lambda]][−] [-Hanahan (1983) J. Mol.Biol. 166, 557-580; XL1Blue-MRF′-D(mcrA)182,D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac,l-, [F′proAB, lac I[q]ZDM15, Tn10 (tet[r])]; SURE Cells [Stratagene];e14(mcrA), D(mcrCB-hsdSMR-mrr)171, sbcC, recB, recJ, umuC::Tn5 (kan[r]),uvrC, supE44, lac, gyrA96, relA1, thi-1, end A1[F′proAB, lacI[q]DM15,Tn10(tet[r])]; GM272-F[−], hsdR544 (rk([−], mk[−]), supE44, supF58,lacY1 or [[Delta]]lacIZY6, galK2, galT22, metB1m, trpR55, [[lambda]][−];HB101-F[−], hsds20 (rb[−], mb[−]), supE44, ara14, galK2, lacY1, proA2,rpsL20 (str[R]), xyl-5, mtl-1, [[lambda]][−], recA13, mcrA(+), mcrB(−)[Raleigh and Wilson (1986) Proc. Natl. Acad. Sci. USA 83, 9070-9074];JM101-supE, thi, [[Delta]](lac-proAB), [F′, traD36, proAB,lacIqZ[[Delta]]M15], restriction: (rk([+], mk([+]), mcrA+[Yanisch-Perron et al. (1985) Gene 33, 103-119]; XL-1 blue recA1, endA1,gyrA96, thi, hsdR17 (rk[+], mk[+]), supE44, relA1, [[lambda]][−], lac,[F′, proAB, lacIqZ[[Delta]]M15, Tn10 (tet[R])][-Bullock, et al. (1987)BioTechniques 5, 376-379]; GM2929 [from B. Bachman, Yale E. coli GeneticStock Center (CSGC#7080)]; M.Marinus strain; sex F[−];(ara-14, leuB6,fhuA13, lacY1, tsx-78, supE44, [glnV44], galK2, galT22, l[−], mcrA,dcm-6, hisG4,[Oc], rfbD1, rpsL136, dam-13::Tn9, xyl-5, mtl-1, recF143,thi-1, mcrB, hsdR2.), MC1000-(araD139, D[ara-leu]7679, gaIU, galK,D[lac]174, rpsL, thi-1); ED8767(F-,e14-[mcrA],supE44,supF58,hsdS3[rB[−]mB[−]], recA56, galK2,galT22,metB1, lac-3 or lac3Y1. Suitable prokaryotic host cells areavailable from the American Type Culture Collection, Manassas, Va., US,other public cell depositaries, and a variety of academic and commercialsources. Selection of a suitable cloning system or cell is not alimitation of the present invention.

Each of the movable cassettes used in the construct of the invention isflanked by a unique set of rare restriction enzyme sites. Each set ofrare restriction enzyme sites provides a first rare restriction enzymesite at the 5′ end of the movable cassette and a second rare restrictionenzyme site the 3′ end of the movable cassette. In one embodiment, theset of rare restriction enzyme sites allows directional cloning of anexpression cassette into the locus. However, the invention is notlimited to the direction of the insert. In other words, the movablecassette and/or the heterologous expression cassette may be locatedeither 5′ to 3′, or 3′ to 5′ with respect to the orientation of thereading frame of the surrounding viral genome. Further, in certainembodiments, the set of rare restriction enzyme sites may allownon-directional cloning of the expression cassette into the selectedlocus.

In one example, the rare restriction enzyme I-SceI may be selected forboth the 5′ and 3′ rare restriction enzymes sites which compose a singleset. This enzyme allows directional cloning, even when flanking bothends of a cassette. In other embodiments, I-SceI may be used incombination with another rare restriction enzyme to form a set ofrestriction enzyme sites. Suitably, each set of rare restriction enzymesis unique, to allow digestion of only a single locus and ready insertionof a heterologous expression cassette into a selected target site.

In a further embodiment, the rare restriction enzyme is selected so thatonly the selected location(s) in the viral genome are cleaved, i.e.,cleavage is achieved at only the 5′ and 3′ ends of the movable cassetteand/or heterologous expression cassette, and neither the genetic elementcarrying the viral genome or other locations in the viral genome arecut.

In the present application, such a restriction enzyme is termed a rarecutter. Examples of such rare cutters include those having recognitionssites for seven, eight, or more bases, including, e.g., I-Ceu I, PI-SceI, TevII, BmoI, DmoI, FseI, PacI, PmeI, PsrI, BcgI, BglI, BsabI, BstXI,DrdI, EcoNI, FseI, MaM I, Msl I, Mwo I, Psha I, Sfi I, Swa I, Xcm I, andXmn I, and the like. Suitable rare cutters may be identified usinginformation readily available to those of skill in the art in theliterature and in a variety of on-line databases, e.g., the REBASE™database. Suitable cutters for the method can be readily determinedusing a variety of computer programs and/or on-line databases. Suitablerestriction enzymes are available from a variety of commercial sourcesincluding, e.g., England Biolabs, Obiogene, Lift Technology, Roche, B BClontech, Stratagene, Amersham Pharmacia, among others.

Thus, the polyvalent plasmid of the invention contains at least twomovable cassettes, each of which is flanked by a unique set of rarerestriction enzymes that permits selective replacement of the movablecassettes with a heterologous expression cassette. These polyvalentplasmids are transfected into host cells that permit expression of themarkers carried by the movable cassettes

B. Polyvalent Transfer Vector Carrying Heterologous Expression Cassettes

Once the appropriate digestion enzyme(s) is selected, conventionaldigestion and religation techniques are utilized. Typically, the plasmidDNA is mixed with the restriction enzyme(s) and incubated for about 12to about 48 hours. Following this, a conventional phenol/chloroformextraction step is performed. For example, phenol/chloroform extractionmay be utilized, followed by precipitation with ethanol, and dissolvingthe precipitate (e.g., in TE or another suitable buffer) for use in theremainder of the method steps. See, e.g., Sambrook, Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., 5.28-5.32, Appendix E.3-E.4 (Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989). Other suitable methodsmay be provided by the manufacturer or vendor of the restriction enzymeutilized, or otherwise known to those of skill in the art.

Typically, in order to ensure proper insertion of the heterologousexpression cassette, the heterologous expression cassettes is flanked atits 5′ and 3′ end with restriction enzyme restriction sites which arecomplementary to the set of restriction enzyme sites that flank themovable cassette for the site in which the expression cassettes areinserted.

Thus, typically, a first heterologous expression cassette is cloned intothe site of the excised movable cassette. Advantageously, the method ofthe invention enables the rapid identification of plasmids containingthe first heterologous expression cassette. Such plasmids lackexpression of the first marker gene, but will be expressing the secondmarker gene product (as well as any other marker gene products present).In other words, if the first movable cassette expressed greenfluorescent protein, the absence of green color following digestion andreligation will indicate successful removal of the movable cassette intothe site of the first marker gene.

In one embodiment, the digestion and religation steps are repeatedsequentially for each of the movable cassettes. In other words, a firstdigestion step is performed using a first set of restriction enzymes toremove a single movable cassette, in order to ensure that theheterologous expression cassette is inserted in to the desired locus.Thereafter, a second digestion step is performed using a second set ofrestriction enzyme unique to the set flanking the second movablecassette. A second heterologous expression cassette flanked byrestriction enzyme sites corresponding to the set flanking the secondmovable cassette is ligated into the plasmid backbone and clones areselected which lack expression of the second marker gene. Optionally,one or more further digestion steps are performed in order to remove oneor more further movable cassettes.

Thus, the method of the invention permits efficient production of apolyvalent transfer vector useful for production of infectious viralparticles.

II. Method of Producing Polyvalent Viral Vector

A polyvalent transfer vector of the invention can be used to generateviral particles, having a capsid or an envelope, using methods known tothose of skill in the art. Such techniques include conventional cloningtechniques of cDNA such as those described in texts [Sambrook et al.,cited above], use of overlapping oligonucleotide sequences of theadenovirus genomes, polymerase chain reaction, and any suitable methodwhich provides the desired nucleotide sequence. Standard transfectionand co-transfection techniques are employed, e.g., CaPO₄ precipitationtechniques. Other conventional methods employed include homologousrecombination of the viral genomes, plaquing of viruses in agar overlay,methods of measuring signal generation, and the like.

Suitable production cell lines are readily selected by one of skill inthe art. For example, a suitable host cell can be selected from anybiological organism, including prokaryotic (e.g., bacterial) cells, andeukaryotic cells, including, insect cells, yeast cells and mammaliancells. Host cells can be selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,HEK 293 cells or PERC6 (both of which express functional adenoviral E1)[Fallaux, F J et al., (1998), Hum Gene Ther, 9:1909-1917], Saos, C2C12,L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblastcells derived from mammals including human, monkey, mouse, rat, rabbit,and hamster. The selection of the mammalian species providing the cellsis not a limitation of this invention; nor is the type of mammaliancell, i.e., fibroblast, hepatocyte, tumor cell, etc.

Generally, when delivering the polyvalent transfer vector comprising theheterologous expression cassettes to a host cell by transfection, thebackbone is delivered in an amount from about 5 μg to about 100 μg DNA,or about 10 to about 50 μg DNA to about 1×10⁴ cells to about 1×10¹³cells, or about 10⁵ cells. However, the relative amounts of plasmid DNAto host cells may be adjusted, talking into consideration such factorsas the selected vector, the delivery method and the host cells selected.

Typically, the polyvalent transfer vectors are cultured in the hostcells which express the capsid protein and/or envelope protein. In thehost cells, the polyvalent viral genomes expressing the heterologousexpression cassettes are rescued and packaged into the capsid protein orenvelope protein to form an infectious viral particle.

A. Viral Vectors Having Capsid Proteins

In one embodiment, the invention provides a method of packaging apolyvalent viral genome into an infectious viral capsid.

In one embodiment, the viral capsid is derived from an adenovirus. Anadenoviral particle or vector of the present invention is composed of aninfectious adenovirus protein capsid having packaged therein apolyvalent viral genome containing two or more heterologous expressioncassettes, each of these cassettes carrying a product to be expressed inthe host. In a further embodiment, these adenoviral vectors arereplication-defective, thereby avoiding replication in a host cell.

The selection of the serotype of the adenoviral sequences present invector is not a limitation of the present invention. A variety ofadenovirus strains are available from the American Type CultureCollection, Manassas, Va., or available by request from a variety ofcommercial and institutional sources. Further, the sequences of manysuch strains are available from a variety of databases including, e.g.,PubMed™ and GenBank™. Homologous adenovirus vectors prepared from othersimian or from human adenoviruses are described in the publishedliterature [see, for example, U.S. Pat. No. 5,240,846]. The DNAsequences of a number of adenovirus types are available from GenBank,including type Ad5 [GenBank™ Accession No. M73260]. The adenovirussequences may be obtained from any known adenovirus serotype, such asserotypes 2, 3, 4, 7, 12 and 40, and further including any of thepresently identified human types. Similarly adenoviruses known to infectnon-human animals (e.g., simians) may also be employed in the vectorconstructs of this invention. In one embodiment, at least one of theadenoviruses used in the invention is derived from a non-human primate.Examples of suitable non-human primate sequences including simianadenoviruses, such as, Pan5 (also C5), Pan6 (also C6), Pan7 (also C7),Pan 9 (also C68) and C1. Recombinant adenoviruses have been describedfor delivery of molecules to host cells. See, U.S. Pat. No. 6,083,716,which provides adenoviral vectors derived from the two chimpanzeeadenoviruses, C1 and C68 (also termed Pan 9) and International PatentPublication No. WO 02/33645 [Pan 5, Pan6, Pan7-derived vectors].However, the invention is not so limited.

A variety of production methods for adenoviral particles is known tothose of skill in the art. The selection of appropriate productionmethods are not a limitation of the present invention. See, e.g., U.S.Pat. No. 6,083,716; International Patent Publication No. WO 02/33645;and U.S. patent application Ser. No. 10/465,302 and its internationalcounterpart, WO 2005/001103.

Briefly, a polyvalent adenoviral transfer vector of the invention thatlacks the ability to express a functional version of any essentialadenoviral gene product (e.g., E1a, E1b, E2a, E2b, and/or E4 ORF6) canbe cultured in the presence of the missing adenoviral gene productswhich are required for viral infectivity and propagation of anadenoviral particle. These helper functions may be provided by culturingthe backbone in the presence of one or more helper constructs (e.g., aplasmid or virus) or a packaging host cell. See, for example, thetechniques described for preparation of a “minimal” human adenovirus(Ad) vector in International Patent Publication No. WO96/13597,published May 9, 1996.

1. Helper Viruses

Thus, depending upon the adenovirus gene content of the polyvalenttransfer vector employed to carry the expression cassettes, a helperadenovirus or non-replicating virus fragment may be necessary to providesufficient adenovirus gene sequences necessary to produce an infectiverecombinant viral particle containing the expression cassette. Usefulhelper viruses contain selected adenovirus gene sequences not present inthe adenovirus vector construct and/or not expressed by the packagingcell line in which the vector is transfected. In one embodiment, thehelper virus is replication-defective and contains a variety ofadenovirus genes in addition to the sequences described above. Such ahelper virus is desirably used in combination with an E1-expressing cellline.

Helper viruses may also be formed into poly-cation conjugates asdescribed in Wu et al., J. Biol. Chem., 264:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J, 299:49 (Apr. 1, 1994). Helper virusmay optionally contain a second reporter expression cassette. A numberof such reporter genes are known to the art. The presence of a reportergene on the helper virus which is different from the gene product on theadenovirus vector allows both the Ad backbone vector and the helpervirus to be independently monitored. This second reporter is used toenable separation between the resulting recombinant virus and the helpervirus upon purification.

2. Complementation Cell Lines

To generate recombinant adenoviruses (Ad) deleted in any of the genesdescribed above, the function of the deleted gene region, if essentialto the replication and infectivity of the virus, must be supplied to therecombinant virus by a helper virus or cell line, i.e., acomplementation or packaging cell line. In many circumstances, a cellline expressing the human E1 can be used to transcomplement the chimp Advector. This is particularly advantageous because, due to the diversitybetween the chimp Ad sequences of the invention and the human AdE1sequences found in currently available packaging cells, the use of thecurrent human E1-containing cells prevents the generation ofreplication-competent adenoviruses during the replication and productionprocess. However, in certain circumstances, it will be desirable toutilize a cell line that expresses the E1 gene products for productionof an E1-deleted simian adenovirus. Such cell lines have been described.See, e.g., U.S. Pat. No. 6,083,716.

If desired, one may utilize the sequences provided herein to generate apackaging cell or cell line that expresses, at a minimum, the adenovirusE1 gene under the transcriptional control of a promoter for expressionin a selected parent cell line. Inducible or constitutive promoters maybe employed for this purpose. Examples of such promoters are describedin detail elsewhere in this specification. A parent cell is selected forthe generation of a novel cell line expressing any desired Ad gene.Without limitation, such a parent cell line may be HeLa [ATCC AccessionNo. CCL 2], A549 [ATCC Accession No. CCL 185], HEK 293, KB [CCL 17],Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75], among others.These cell lines are all available from the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209. Othersuitable parent cell lines may be obtained from other sources.

Such E1-expressing cell lines are useful in the generation ofrecombinant adenovirus E1 deleted vectors. Additionally, oralternatively, the invention provides cell lines that express one ormore simian adenoviral gene products, e.g., E1a, E1b, E2a, and/or E4ORF6, can be constructed using essentially the same procedures for usein the generation of recombinant simian viral vectors. Such cell linescan be utilized to transcomplement adenovirus vectors deleted in theessential genes that encode those products. The preparation of a hostcell according to this invention involves techniques such as assembly ofselected DNA sequences. This assembly may be accomplished utilizingconventional techniques. Such techniques include cDNA and genomiccloning, which are well known and are described in Sambrook et al.,cited above, use of overlapping oligonucleotide sequences of theadenovirus genomes, combined with polymerase chain reaction, syntheticmethods, and any other suitable methods which provide the desirednucleotide sequence.

In still another alternative, the essential adenoviral gene products areprovided in trans by a vector and/or helper virus. In such an instance,a suitable host cell can be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. Suitable hostcells include those known in the art, as well as those identifiedherein.

3. Assembly of Viral Particle and Transfection of a Cell Line

One or more of the missing adenoviral genes may be stably integratedinto the genome of the host cell, stably expressed as episomes, orexpressed transiently. The gene products may all be expressedtransiently, on an episome or stably integrated, or some of the geneproducts may be expressed stably while others are expressed transiently.

Furthermore, the promoters for each of the adenoviral genes may beselected independently from a constitutive promoter, an induciblepromoter or a native adenoviral promoter. The promoters may beregulated, for example, by a specific physiological state of theorganism or cell (i.e., by the differentiation state or in replicatingor quiescent cells) or by exogenously-added factors. Introduction of themolecules (as plasmids or viruses) into the host cell may also beaccomplished using techniques known to the skilled artisan and asdiscussed throughout the specification. In one embodiment, directcloning techniques are utilized. Such techniques have been described [G.Gao et al., Gene Ther. 2003 October; 10(22):1926-1930; US PatentPublication No. 2003-0092161-A, May 15, 2003; International PatentApplication No. PCT/US03/12405]. In another embodiment, standardtransfection techniques are used, e.g., CaPO₄ transfection orelectroporation. Assembly of the selected DNA sequences of theadenovirus (as well as the sequences encoding the product and othervector elements) into various intermediate plasmids, and the use of theplasmids and vectors to produce a recombinant viral particle are allachieved using conventional techniques. For example, following theconstruction and assembly of the desired polyvalent viral vector, thevector is transfected in vitro in the presence of a helper virus intothe packaging cell line. Homologous recombination occurs between thehelper and the vector sequences, which permits the adenovirus-genesequences in the vector to be replicated and packaged into virioncapsids, resulting in the recombinant viral vector particles. Thecurrent method for producing such virus particles is transfection-based.However, the invention is not limited to such methods.

The resulting recombinant adenoviruses are useful in transferring two ormore selected heterologous expression cassettes to a selected cell.

B. Viral Vectors Having Envelope Proteins

In another embodiment, the transfer vectors of the invention are used topackage a viral vector into an infectious particle of a virus having anenvelope protein, e.g, a lentivirus or a poxvirus. Examples of suitablelentiviruses include, e.g., human immunodeficiency virus (HIV), simianimmunodeficiency virus (SIV), caprine arthritis and encephalitis virus,equine infectious anemia virus, bovine immunodeficiency virus, visnavirus, and feline immunodeficiency virus (FIV). The examples providedherein illustrate the use of minigenes derived from HIV and FIV.However, other lentiviruses of human or non-human origin may also beused. The sequences used in the constructs of the invention may bederived from academic, non-profit (e.g., the American Type CultureCollection, Manassas, Va.) or commercial sources of lentiviruses.

Methods of generating such viral vectors are known to those of skill inthe art.

Methods of producing lentiviral vectors have been described. See, e.g.,J E Coleman, et al., Physiol Genomics. 2003 Feb. 6; 12(3):221-8., whichdescribed production of an HIV-1 based lentiviral system usingself-inactivating lentiviral vectors. In one example, lentiviral vectorsare created in a transient transfection system in which a cell line istransfected with three separate plasmid expression systems. Theseinclude the transfer vector plasmid, which is produced according to thepresent invention and contains portions of the selected lentiviralprovirus and the heterologous expression cassettes, the packagingplasmid or construct, and a plasmid with a lentiviral envelope gene(ENV) which may be an envelope protein of the same lentivirus or adifferent virus [Amado and Chen, Lentiviral Vectors—the Promise of GeneTherapy Within Reach? Science. 285 (5428): 674-76 (1999)]. The threeplasmid components of the vector are put into a packaging cell which isthen inserted into a lentiviral shell.

In one embodiment, the transfer vector plasmid contains cis-actinggenetic sequences necessary for the vector to infect the target cell andfor transfer of the therapeutic (or reporter) gene and containsrestriction sites for insertion of desired genes. The 3′ and 5′ LTRs,the original envelope proteins, and gag sequence promoter have beenremoved. The packaging plasmid contains the elements required for vectorpackaging such as structural proteins, HIV genes (except the gene envwhich codes for infection of T cells, or the vector would only be ableto infect these cells), and the enzymes that generate vector particles.Typically, the packaging signals and their adjacent signals are removedso the parts responsible for packaging the viral DNA have been separatedfrom the parts that activate them. Thus, the packaging sequences willnot be incorporated into the viral genome and the virus will notreproduce after it has infected the host cell.

The third plasmid's envelope gene of a different virus specifies whattype of cell to target and infect instead of the T cells, e.g., theglycoprotein of vesicular stomatitis virus, known as VSV, MLV, amongothers. Normally HIV can infect only helper T-cells because they usetheir gp120 protein to bind to the CD4 receptor. However, it is possibleto genetically exchange the CD4 receptor-binding protein for anotherprotein that codes for the different cell type on which gene therapywill be performed. This gives the lentiviral vector a broad range ofpossible target cells. Other lentiviral production methods and vectorelements are described in International Patent Publication No. WO03/092582, which is incorporated by reference.

Still other enveloped viral vectors can be produced using the polyvalentviral backbones methods of the invention. See, e.g. “The Uses ofPoxviruses as Vectors”, Current Gene Therapy, vol. 3, no. 6, pp. 583-595(December 2003); M. E. Perkus, et al., “Poxvirus-based vaccinecandidates for cancer, AIDS, and other infectious diseases”, Journal ofLeukocyte Biology, Vol 58, Issue 1 1-13, (1995).

III. Uses for Polyvalent Viral Vector

The polyvalent viral vectors of the invention are formulated in acomposition containing a physiologically compatible carrier. Thesecompositions are useful in a variety of therapeutic and immunizationregimens. Advantageously, the expression of multiple gene products fromthe polyvalent viral vectors may reduce the amount of vector, or otherdrug, necessary to deliver to the subject to achieve the desiredbiological effect.

In one embodiment, the polyvalent viral vector of the invention containsa heterologous expression cassette carrying a therapeutic product. Sucha polyvalent viral vector can further carry one or more additionaltherapeutic products. An additional therapeutic product can be directedto treatment the same conditions or symptoms related to the firsttherapeutic product, or to a different indication. Where desired, aselected therapeutic gene product can be delivered to modulate anyreaction to the polyvalent viral vector.

In another embodiment, the polyvalent viral vector of the inventioncontains a heterologous expression cassette carrying a product whichinduces a humoral and/or cytotoxic immune response to a target. Such apolyvalent viral vector can further carry one or more additional suchimmunogenic products. This second product can be directed to inducing animmune response to the target, or a cross-reactive target. In anotherembodiment, the second product can be a therapeutic product designed totreat symptoms associated with the underlying condition. In stillanother embodiment, the second product can be an adjuvant for anothergene product delivered by the polyvalent viral vector. In yet anotherembodiment, the second product is a therapeutic product delivered tomodulate any reaction to the polyvalent viral vector.

As used herein, immune modulators include products that modify thereaction of the immune system, e.g., to a viral vector. Examples ofimmune modulators include, e.g., cytokines and interleukins.

As used herein, suitable immunomodulatory compounds can include, e.g.,CTLA4 immunoglobulin; anti-CD4 antibodies; FK506; and interleukins (IL),including any of IL1-21, e.g., IL-2, IL-3, IL-4, IL-10, IL-12, andIL-18. For example, IL-10 may be useful in down-modulating a localanti-inflammatory response; Fas ligand may be useful in down-regulatingadenovirus-mediated T cell responses.

Still other suitable combinations of products to be delivered in apolyvalent viral vector of the invention, in a cocktail containing oneor more polyvalent viral vectors of the invention, or in a regimeninvolving delivery of one or more polyvalent viral vectors of theinvention, will be apparent to those of skill in the art from thepresent specification.

A. Polyvalent Viral Vector-Mediated Delivery of Therapeutic Molecules

In one embodiment, the polyvalent vectors are administered to humansaccording to published methods for gene therapy. A viral polyvalentvector carrying multiple heterologous expression control cassettes maybe administered to a patient, preferably suspended in a biologicallycompatible solution or pharmaceutically acceptable delivery vehicle. Asuitable vehicle includes sterile saline. Other aqueous and non-aqueousisotonic sterile injection solutions and aqueous and non-aqueous sterilesuspensions known to be pharmaceutically acceptable carriers and wellknown to those of skill in the art may be employed for this purpose.

The polyvalent vectors are administered in sufficient amounts totransduce the target cells and to provide sufficient levels of genetransfer and expression to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the retina and other intraoculardelivery methods, direct delivery to the liver, inhalation, intranasal,intravenous, intramuscular, intratracheal, subcutaneous, intradermal,rectal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe gene product or the condition. The route of administration primarilywill depend on the nature of the condition being treated.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectiveadult human or veterinary dosage of the viral vector is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² particles virus.Dosages will range depending upon the size of the animal and the routeof administration. For example, a suitable human or veterinary dosage(for about an 80 kg animal) for intramuscular injection is in the rangeof about 1×10⁹ to about 5×10¹² particles per mL, for a single site.Optionally, multiple sites of administration may be delivered. Inanother example, a suitable human or veterinary dosage may be in therange of about 1×10¹¹ to about 1×10¹⁵ particles for an oral formulation.One of skill in the art may adjust these doses, depending the route ofadministration, and the therapeutic or vaccinal application for whichthe recombinant vector is employed. The levels of expression of thetherapeutic product, or for an immunogen, the level of circulatingantibody, can be monitored to determine the frequency of dosageadministration. Yet other methods for determining the timing offrequency of administration will be readily apparent to one of skill inthe art.

In one embodiment, a polyvalent viral vector contains a heterologousexpression cassette encoding a therapeutic product and a heterologousexpression cassette encoding an immune modulator. The selected immunemodulator is defined herein as an agent capable of inhibiting theformation of neutralizing antibodies directed against the recombinantvector of this invention or capable of inhibiting cytolytic T lymphocyte(CTL) elimination of the vector. The immune modulator may interfere withthe interactions between the T helper subsets (T_(H1) or T_(H2)) and Bcells to inhibit neutralizing antibody formation. Alternatively, theimmune modulator may inhibit the interaction between T_(H1) cells andCTLs to reduce the occurrence of CTL elimination of the vector. Avariety of useful immune modulators and dosages for use of same aredescribed, for example, in Yang et al., J. Virol., 70(9) (September,1996); International Patent Publication No. WO 96/12406, published May2, 1996; and International Patent Publication No. WO 96/26285.

1. Therapeutic Product

Useful therapeutic products encoded by the heterologous expressioncassette include hormones and growth and differentiation factorsincluding, without limitation, insulin, glucagon, growth hormone (GH),parathyroid hormone (PTH), growth hormone releasing factor (GRF),follicle stimulating hormone (FSH), luteinizing hormone (LH), humanchorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF),angiopoietins, angiostatin, granulocyte colony stimulating factor(GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF),basic fibroblast growth factor (bFGF), acidic fibroblast growth factor(aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin growth factors I andII (IGF-I and IGF-II), any one of the transforming growth factorsuperfamily, including TGF, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15, any one of theheregluin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other useful gene products include proteins that regulate the immunesystem including, without limitation, cytokines and lymphokines such asthrombopoietin (TPO), interleukins (IL) IL-1 through IL-25 (including,e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractant protein,leukemia inhibitory factor, granulocyte-macrophage colony stimulatingfactor, Fas ligand, tumor necrosis factors and, interferons, and, stemcell factor, flk-2/flt3 ligand. Gene products produced by the immunesystem are also useful in the invention. These include, withoutlimitation, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimericimmunoglobulins, humanized antibodies, single chain antibodies, T cellreceptors, chimeric T cell receptors, single chain T cell receptors,class I and class II MHC molecules, as well as engineeredimmunoglobulins and MHC molecules. Useful gene products also includecomplement regulatory proteins such as complement regulatory proteins,membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1,CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation, including the low density lipoprotein (LDL)receptor, high density lipoprotein (HDL) receptor, the very low densitylipoprotein (VLDL) receptor, and the scavenger receptor. The inventionalso encompasses gene products such as members of the steroid hormonereceptor superfamily including glucocorticoid receptors and estrogenreceptors, Vitamin D receptors and other nuclear receptors. In addition,useful gene products include transcription factors such as jun, fos,max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD andmyogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3,ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins,interferon regulation factor (IRF-1), Wilms tumor protein, ETS-bindingprotein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkheadfamily of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VIII, factor IX, cystathione beta-synthase, branched chainketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionylCoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target. Reduction and/or modulation ofexpression of a gene are particularly desirable for treatment ofhyperproliferative conditions characterized by hyperproliferating cells,as are cancers and psoriasis. Target polypeptides include thosepolypeptides which are produced exclusively or at higher levels inhyperproliferative cells as compared to normal cells. Target antigensinclude polypeptides encoded by oncogenes such as myb, myc, fyn, and thetranslocation gene bcr/abl, ras, src, P53, neu, trk and EGRF. Inaddition to oncogene products as target antigens, target polypeptidesfor anti-cancer treatments and protective regimens include variableregions of antibodies made by B cell lymphomas and variable regions of Tcell receptors of T cell lymphomas which, in some embodiments, are alsoused as target antigens for autoimmune disease. Other tumor-associatedpolypeptides can be used as target polypeptides such as polypeptideswhich are found at higher levels in tumor cells including thepolypeptide recognized by monoclonal antibody 17-1A and folate bindingpolypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce self-directed antibodies. T cellmediated autoimmune diseases include rheumatoid arthritis (RA), multiplesclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Each of these diseases is characterized by T cellreceptors (TCRs) that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases.

The polyvalent viral vectors of the invention are particularly wellsuited for therapeutic regimens in which multiple viral-mediateddeliveries of gene products is desired, e.g., in regimens involvingredelivery of the same product or in combination regimens involvingdelivery of other genes products.

B. Polyvalent Viral Mediated Delivery of Immunogenic Gene Products

The polyvalent viral vectors of the invention may also be employed asimmunogenic compositions. As used herein, an immunogenic composition isa composition to which a humoral (e.g., antibody) or cellular (e.g., acytotoxic T cell) response is mounted to a product delivered by theimmunogenic composition following delivery to a mammal, and preferably aprimate. The present invention provides a polyvalent viral vector thatcan contain in any of its adenovirus sequence deletions a firstheterologous expression cassette encoding a desired immunogen. Thepolyvalent viral vector can further contain a heterologous expressioncassette encoding an adjuvant for the immunogen, additional immunogenicproducts, a therapeutic product, or a product that down-regulates animmune response to the polyvalent viral vector.

When the polyvalent viral vector is a polyvalent adenoviral vector,suited for use as a live recombinant virus vaccine in different animalspecies compared to an adenovirus of human origin, but is not limited tosuch a use. The recombinant polyvalent viruses can be used asprophylactic or therapeutic vaccines against any pathogen for which theantigen(s) crucial for induction of an immune response and able to limitthe spread of the pathogen has been identified and for which the cDNA isavailable.

Such vaccinal (or other immunogenic) compositions are formulated in asuitable delivery vehicle, as described above. Generally, doses for theimmunogenic compositions are in the range defined above for therapeuticcompositions. The levels of immunity of the selected gene can bemonitored to determine the need, if any, for boosters. Following anassessment of antibody titers in the serum, optional boosterimmunizations may be desired.

Optionally, a vaccinal composition of the invention may be formulated tocontain other components, including, e.g. adjuvants, stabilizers, pHadjusters, preservatives and the like. Such components are well known tothose of skill in the vaccine art. Examples of suitable adjuvantsinclude, without limitation, liposomes, alum, monophosphoryl lipid A,and any biologically active factor, such as cytokine, an interleukin, achemokine, a ligands, and optimally combinations thereof. Certain ofthese biologically active factors can be expressed in vivo, e.g., via aplasmid or viral vector. For example, such an adjuvant can beadministered with a priming DNA vaccine encoding an antigen to enhancethe antigen-specific immune response compared with the immune responsegenerated upon priming with a DNA vaccine encoding the antigen only.

The polyvalent viral vectors are administered in a “an immunogenicamount”, that is, an amount of polyvalent viral vector is effective in aroute of administration to transfect the desired cells and providesufficient levels of expression of the selected gene to induce an immuneresponse. Where protective immunity is provided, the polyvalent viralvectors are considered to be vaccine compositions useful in preventinginfection and/or recurrent disease.

Alternatively, or in addition, the vectors of the invention may containa gene encoding a peptide, polypeptide or protein which induces animmune response to a selected immunogen. The polyvalent adenoviralvectors of this invention are expected to be highly efficacious atinducing cytolytic T cells and antibodies to the inserted heterologousantigenic protein expressed by the vector.

For example, immunogens may be selected from a variety of viralfamilies. Examples of viral families against which an immune responsewould be desirable include, the picornavirus family, which includes thegenera rhinoviruses, which are responsible for about 50% of cases of thecommon cold; the genera enteroviruses, which include polioviruses,coxsackieviruses, echoviruses, and human enteroviruses such as hepatitisA virus; and the genera apthoviruses, which are responsible for foot andmouth diseases, primarily in non-human animals. Within the picornavirusfamily of viruses, target antigens include the VP1, VP2, VP3, VP4, andVPG. Another viral family includes the calcivirus family, whichencompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Other target antigens may be generated from theHepatitis C [see, e.g., US Published Patent Application No. US2003/190606 (Oct. 9, 2003); US 2002/081568 (Jun. 27, 2002)] or thecoronavirus family, which includes a number of non-human viruses such asinfectious bronchitis virus (poultry), porcine transmissiblegastroenteric virus (pig), porcine hemagglutinating encephalomyelitisvirus (pig), feline infectious peritonitis virus (cats), feline entericcoronavirus (cat), canine coronavirus (dog), and human respiratorycoronaviruses, which may cause the common cold and/or non-A, B or Chepatitis, and the putative causative agent of sudden acute respiratorysyndrome (SARS). Within the coronavirus family, target antigens includethe E1 (also called M or matrix protein), E2 (also called S or Spikeprotein), E3 (also called HE or hemagglutin-elterose) glycoprotein (notpresent in all coronaviruses), or N (nucleocapsid). Still other antigensmay be targeted against the rhabdovirus family, which includes thegenera vesiculovirus (e.g., Vesicular Stomatitis Virus), and the generallyssavirus (e.g., rabies). Within the rhabdovirus family, suitableantigens may be derived from the G protein or the N protein. The familyfiloviridae, which includes hemorrhagic fever viruses such as Marburgand Ebola virus, may be a suitable source of antigens. The paramyxovirusfamily includes parainfluenza Virus Type 1, parainfluenza Virus Type 3,bovine parainfluenza Virus Type 3, rubulavirus (mumps virus),parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastledisease virus (chickens), rinderpest, morbillivirus, which includesmeasles and canine distemper, and pneumovirus, which includesrespiratory syncytial virus. The influenza virus is classified withinthe family orthomyxovirus and is a suitable source of antigen (e.g., theHA protein, the N1 protein). The bunyavirus family includes the generabunyavirus (California encephalitis, La Crosse), phlebovirus (RiftValley Fever), hantavirus (puremala is a hemahagin fever virus),nairovirus (Nairobi sheep disease) and various unassigned bungaviruses.The arenavirus family provides a source of antigens against LCM andLassa fever virus. The reovirus family includes the genera reovirus,rotavirus (which causes acute gastroenteritis in children), orbiviruses,and cultivirus (Colorado Tick fever, Lebombo (humans), equineencephalosis, blue tongue).

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp41, or smaller fragments thereof,e.g., of at least about 8 amino acids in length. Similarly, fragments ofthe tat protein may be selected. [See, U.S. Pat. No. 5,891,994 and U.S.Pat. No. 6,193,981.] See, also, the HIV and SIV proteins described in D.H. Barouch et al., J. Virol., 75(5):2462-2467 (March 2001), and R. R.Amara, et al., Science, 292:69-74 (6 Apr. 2001). In another example, theHIV and/or SIV immunogenic proteins or peptides may be used to formfusion proteins or other immunogenic molecules. See, e.g., the HIV-1 Tatand/or Nef fusion proteins and immunization regimens described inInternational Patent Publication No. WO 01/54719, published Aug. 2,2001, and International Patent Publication No WO 99/16884, publishedApr. 8, 1999. The invention is not limited to the HIV and/or SIVimmunogenic proteins or peptides described herein. In addition, avariety of modifications to these proteins has been described or couldreadily be made by one of skill in the art. See, e.g., the modified gagprotein that is described in U.S. Pat. No. 5,972,596. Further, anydesired HIV and/or SIV immunogens may be delivered alone or incombination. Such combinations may include expression from a singlevector or from multiple vectors.

Optionally, another combination may involve delivery of one or moreexpressed immunogens with delivery of one or more of the immunogens inprotein form. Such combinations are discussed in more detail below. Thepapovavirus family includes the sub-family polyomaviruses (BKU and JCUviruses) and the sub-family papillomavirus (associated with cancers ormalignant progression of papilloma). For example, papillomavirusantigens and combinations thereof have been described. See, e.g., USPublished Application No. 2003/129199 (Jul. 10, 2003); US PublishedApplication No. 2002/18221 (Dec. 15, 2002); U.S. Pat. No. 6,342,224.

The adenovirus family includes viruses (EX, AD7, ARD, O.B.) which causerespiratory disease and/or enteritis. The parvovirus family felineparvovirus (feline enteritis), feline panleucopeniavirus, canineparvovirus, and porcine parvovirus. The herpesvirus family includes thesub-family alphaherpesvirinae, which encompasses the genera simplexvirus(HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster) and thesub-family betaherpesvirinae, which includes the genera cytomegalovirus(HCMV, muromegalovirus) and the sub-family gammaherpesvirinae, whichincludes the genera lymphocryptovirus, EBV (Burkitts lymphoma),infectious rhinotracheitis, Marek's disease virus, and rhadinovirus. Thepoxvirus family includes the sub-family chordopoxyirinae, whichencompasses the genera orthopoxvirus (Variola (Smallpox) and Vaccinia(Cowpox)), parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus,suipoxvirus, and the sub-family entomopoxyirinae. The hepadnavirusfamily includes the Hepatitis B virus. One unclassified virus which maybe suitable source of antigens is the Hepatitis delta virus. Still otherviral sources may include avian infectious bursal disease virus andporcine respiratory and reproductive syndrome virus. The alphavirusfamily includes equine arteritis virus and various Encephalitis viruses.

The present invention may also encompass immunogens which are useful toimmunize a human or non-human animal against other pathogens includingbacteria, fungi, parasitic microorganisms or multicellular parasiteswhich infect human and non-human vertebrates, or from a cancer cell ortumor cell. Examples of bacterial pathogens include pathogenicgram-positive cocci include pneumococci; staphylococci; andstreptococci. Pathogenic gram-negative cocci include meningococcus;gonococcus. Pathogenic enteric gram-negative bacilli includeenterobacteriaceae; pseudomonas, acinetobacteria and eikenella;melioidosis; salmonella; shigella; haemophilus; moraxella; H. ducreyi(which causes chancroid); brucella; Franisella tularensis (which causestularemia); yersinia (pasteurella); streptobacillus moniliformis andspirillum; Gram-positive bacilli include listeria monocytogenes;erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria);cholera; B. anthracis (anthrax); donovanosis (granuloma inguinale); andbartonellosis. Diseases caused by pathogenic anaerobic bacteria includetetanus; botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever, Rocky Mountain spottedfever, Q fever, and Rickettsialpox. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes encompass pathogenic protozoans and helminths and infectionsproduced thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans;Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm)infections.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the vectors of the invention to deliver immunogensagainst the variable region of the T cells elicit an immune responseincluding cytotoxic T-lymphocytes (CTLs) to eliminate those T cells. Inrheumatoid arthritis (RA), several specific variable regions of T-cellreceptor (TCRs) which are involved in the disease have beencharacterized. These TCRs include V-3, V-14, V-17 and Vα-17. Thus,delivery of a nucleic acid sequence that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in RA. In multiple sclerosis (MS), several specific variableregions of TCRs which are involved in the disease have beencharacterized. These TCRs include V-7 and Vα-10. Thus, delivery of anucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-6,V-8, V-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12.Thus, delivery of a recombinant simian adenovirus that encodes at leastone of these polypeptides will elicit an immune response that willtarget T cells involved in scleroderma.

The following examples illustrate the cloning of the polyvalentadenoviruses and the construction of exemplary polyvalent adenovirusvectors of the present invention. These examples are illustrative only,and do not limit the scope of the present invention.

EXAMPLES

As illustrated if FIGS. 1 and 2, chimpanzee adenovirus Pan9 based vectoris constructed as a polyvalent transfer vector according to theinvention.

A. Construction of DNA Molecule with Movable Cassettes

With reference to FIG. 1, a plasmid backbone was selected which carriesthe chimpanzee adenovirus Pan9 viral genome, having a mutant greenfluorescent protein marker (GFP) cassette in the site of the Pan9 E1gene region, the bovine growth hormone polyA, and a partial E3-deletion.This plasmid is described in more detail in International PatentPublication No. WO 2003/046124.

pPan9-pkGFP is digested with AvrII (FIG. 1A) to remove the E3 region ofPan 9 which is cloned into pSL1180 [obtained commercially fromPharmacia], to form pSL1180-Pan9-Avr(II), which contains the ampicillinresistance gene.

Plasmid RSV-Red2 contains an RSV promoter, a lac promoter driving theAsRed2 product (Lac-AsRed2), flanked by an I-SceI and PI-PspI site,followed by an SV40 PolyA. This pRSV-Red2 plasmid was constructed bycloning the RSV promoter into a plasmid constructed from pUC19 and pkRFP(Clontech). The pSL1180-Pan9-Avr(II) plasmid was digested by NruI (FIG.1B) to release the Pan9 E3 fragment, pRSV-Red2 was digested with PvuIIand HpaI (FIG. 1C) to release the I-SceI-RSV-lac-AsRed2-PI-SceI cassetteand religated to form a plasmid SL1180-Pan9-Avr(II)pkRFP, which containsthe ampicillin resistance gene, and the I-SceI-RSV-lac-AsRed2-PI-SceIcassette in the site of the E3 region.

Also resulting from the AvrII digestion of pPan9-pkGFP described aboveis a plasmid containing the Pan9 viral genome having a deletion in theE3 region (FIG. 1D); following religation, the resulting plasmid istermed pPan9-pkGFP-Avr(II). This plasmid and thepSL1180-Pan9-Avr(II)pkRFP plasmid are digested with AvrII (FIG. 1E) andreligated to form a single plasmid containing the GFP reporter gene inthe E1 locus and the RFP reporter gene in the E3 locus. The resultingplasmid is termed pPan9-(E1)-pkGFP-(E3)pkRFP. By selecting for coloniesexpressing yellow, one selects for vectors expressing both the green andred fluorescent proteins.

This design allows easy shuttle in the antigen expression cassettes andconvenient color-based colony selection of recombinants, which will besuitable for high throughput vector creation.

B. Construction of Polyvalent Transfer Vector

In order to construct a polyvalent transfer vector of the invention, afirst selected heterologous expression cassette is cloned into anappropriate site in a shuttle vector.

With reference to FIG. 2, this is illustrated by cloning “antigene 1”into a plasmid having multiple cloning site, selecting the site flankedby restriction enzyme sites corresponding to those in the desired regionof the DNA molecule produced according to part A above, i.e., flanked byI-CeuI and PI-SceI sites.

A second heterologous expression cassette (antigene 2) is cloned into anappropriate vector (e.g., pUC19-RSV) between I-SceI sites.

Following digestion with the appropriate enzymes, vectors containing thedesired heterologous expression cassettes can be selected bycolorimetric means. More particularly, colonies expressing red uponexcitation with the appropriate wavelength of light indicate thepresence of vectors in which the movable cassette containing the GFPhave been replaced with antigene 1, but the movable cassette containingthe RFP has been retained. Colonies expressing green upon excitationwith the appropriate wavelength of light indicate the presence ofvectors in which the movable cassette containing the RFP has beenreplaced with antigene 1, but the movable cassette containing the GFPhas been retained. Colonies which appear white after excitation withlight of the appropriate wavelengths for the GFP and RFP indicatevectors in which both movable cassettes have been replaced withheterologous expression cassettes. Thus, by selecting for the whitecolonies, one can rapidly and accurately select for polyvalent transfervectors of the invention.

C. Study of Impact of Location (E1 or E3) and Orientation of TransgeneCassettes on Antigen Expression

Identical CMV-hA1AT expression cassettes were cloned into E1 locus ofE1-deleted Pan9 (also termed C9) vector and E3 locus of E1/E3-deleted C9vector separately. The recombinant C9 viruses expressing A1AT fromdifferent loci were produced and injected into C57BL/6 miceintravenously at a dose of 1×10¹¹ pts/mouse. The animals were bled atdays 3 and 7 post gene transfer and serum hA1AT levels were measured forcomparison. The data revealed that hA1AT expressed from E3 locus wereactually at least 2 folds higher than that from E1 locus. See, FIG. 3.

The cloning process of the invention has a convenient dual colorselection system for the recombinants of mono- or polyvalent vector.Further, this data demonstrates that there is no negative locusdependent effect on the transgene expression in simian adenovirusvectors.

All publications cited in this specification, and the sequence listing,are incorporated herein by reference. While the invention has beendescribed with reference to particular embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. A polyvalent plasmid backbone comprising: (a) a first movablecassette located in a first locus of a viral genome, said first movablecassette comprising nucleic acid sequences comprising a first detectablereporter gene operably linked to sequences that will direct expressionthereof, said movable cassette being flanked by a first set of rarerestriction enzyme sites composed of two rare restriction enzyme sites;and (b) a second movable cassette located in a second locus of the viralgenome, said second movable cassette comprising nucleic acid sequencescomprising a second detectable reporter gene operably linked tosequences that will direct expression thereof, said movable cassettebeing flanked by a second set of rare restriction enzyme sites composedof two rare restriction enzyme sites; wherein the product of said firstdetectable reporter gene and the second detectable reporter gene aredistinguishable, and wherein the first and second set of rarerestriction enzyme sites differ.
 2. The polyvalent plasmid backboneaccording to claim 1, further comprising three or more loci.
 3. Theplasmid backbone according to claim 1, wherein the first and secondlocus are located in different gene regions within the viral genome. 4.The plasmid backbone according to claim 1, wherein the first and secondlocus are located in different open reading frames within a single generegion of the viral genome.
 5. The plasmid backbone according to claim1, wherein the first and second locus are located within a single openreading frame of a gene region of the viral genome and arenon-contiguous with one another.
 7. The plasmid backbone according toclaim 1, wherein the detectable reporter genes are differentiated fromone another by product color.
 8. The plasmid backbone according to claim1, wherein the detectable reporter genes are independently selected fromthe group consisting of green fluorescent protein, red fluorescentprotein, and beta-galactosidase.
 9. The plasmid backbone according toclaim 1, wherein the viral genome is an adenoviral genome.
 10. Theplasmid backbone according to claim 1, wherein the viral genome isselected from the group consisting of a lentiviral genome, retroviralgenome, and a poxvirus genome.
 11. The plasmid backbone according toclaim 1, wherein the two rare restriction enzyme sites in the first setare independently selected from the group consisting of I-Ceu I, PI-SceI, TevII, BmoI, and DmoI, and differ from one another.
 12. (canceled)13. (canceled)
 14. The method according to claim 13, wherein the firstdetectable reporter gene is green fluorescent protein and the seconddetectable reporter gene is red fluorescent protein.
 15. The methodaccording to claim 13, wherein the first set of enzymes is I-Ceu I andPI-Sce I.
 16. The method according to claim 13, wherein the second setof enzymes differs from the first set of enzymes.
 17. The methodaccording to claim 13, wherein the polyvalent transfer vector comprisessequences from a viral genome selected from the group consisting of anadenovirus, a lentivirus, a retrovirus, a poxvirus.
 18. The methodaccording to claim 16, wherein the polyvalent transfer vector comprisesadenoviral sequences in which the first expression cassette is locatedin an adenovirus E1 region.
 19. The method according to claim 16,wherein the polyvalent transfer vector comprises adenoviral sequences inwhich the second expression cassette is located in an adenovirus E4region.
 20. The method according to claim 16, wherein the polyvalenttransfer vector comprises adenoviral sequences in which the secondexpression cassette is located in the adenovirus E3 region. 21.(canceled)
 22. (canceled)
 23. A method of generating a polyvalent viruscomprising the step of culturing the polyvalent transfer vector preparedaccording to claim 36 in the presence of sufficient viral sequences topermit packaging of the polyvalent viral genome into an infectious viralenvelope.
 24. A polyvalent virus produced according to the method ofclaim
 23. 25. The polyvalent virus according to claim 24, wherein saidvirus is packaged in an infectious envelope comprising a lentivirusenvelope protein, a retrovirus envelope protein, and a poxvirus envelopeprotein.
 26. The polyvalent virus according to claim 25, wherein thepoxvirus is canarypox.
 27. A method of generating a polyvalent viruscomprising the step of culturing the polyvalent plasmid backboneaccording to claim 13 in the presence of sufficient viral sequences topermit packaging of the polyvalent viral backbone into an infectiousviral capsid.
 28. A polyvalent viral vector produced according to themethod of claim
 27. 29. A polyvalent viral vector according to claim 28comprising an adenoviral capsid protein.
 30. A composition containing:(a) a polyvalent viral vector comprising: (i) a first heterologousexpression cassette comprising a nucleic acid sequence encoding a firsttarget product under the control of regulatory sequence that controlexpression of the product; and (ii) a second heterologous expressioncassette comprising a nucleic acid sequence encoding a second targetproduct under the control of regulatory sequence that control expressionof the product; wherein said first and second expression cassettes arelocated in distinct loci of a viral vector genome and the targetproducts are independently expressed; and (b) a physiologicallycompatible carrier.
 31. The composition according to claim 30, whereinsaid first and second products are independently selected from the groupconsisting of an antigen and a cytokine.
 32. The composition accordingto claim 30, wherein the first or second product is an immune modulator.33. The composition according to claim 32, wherein the second product isan adjuvant for the first product.
 34. The composition according toclaim 32, wherein the first or second product is a therapeutic geneproduct.
 35. A cell culture comprising cells containing the polyvalentplasmid backbone according to claim
 1. 36. A method for generating apolyvalent transfer vector, said method comprising the steps of: (a)mixing, in the presence of the first set of enzymes, a polyvalentplasmid backbone according to claim 1 and a first nucleic acid moleculecomprising a first heterologous expression cassette comprising a nucleicacid sequence encoding a target product under the control of regulatorysequence that control expression of the product, wherein saidheterologous expression cassette is flanked by the first set ofrestriction enzyme sites; (b) selecting for the absence of the firstdetectable reporter to provide plasmid backbones containing the firstheterologous expression cassette; (c) mixing, in the presence of thesecond set of enzymes, the polyvalent plasmid backbone and a secondnucleic acid molecule comprising a second heterologous expressioncassette comprising a nucleic acid sequence encoding a target productunder the control of regulatory sequence that control expression of theproduct, wherein said heterologous expression cassette is flanked by thesecond set of rare restriction enzyme sites; and (d) selecting for theabsence of the second detectable reporter to provide plasmid backbonescontaining the second heterologous expression cassette, therebyproviding a polyvalent transfer vector containing a polyvalent viralgenome comprising a first expression cassette in a first locus and asecond expression cassette in a second locus.
 37. A polyvalent transfervector produced according to the method of claim 13.